In article <6f3h0b$1pa$1 at news1.fast.net>, "Rob Latch" <rlatch at fast.net> wrote:
> I am an undergraduate who recently discovered a bacteriophage. I believe it
> is single stranded, through the use of Mung bean nuclease and restriction
> enzyme analysis. The DNA seems to stain very well with EtBr, and so I am
> wondering if there may be any other tests I may perform that will reassure
> that my phage is single-stranded. Secondly, I'm kind of lost from here. I
> would like to work with this phage, but I am unsure of how to go about it.
> It seems that to do most of the things that may give me additional
> information about my phage, I need to cut it with restriction enzymes.
> However, I can't cut this DNA with any restriction enzymes except for Hha I
> and Mnl I, which shred the DNA. I am looking for any and all protocols that
> would help me to characterize this phage that can be used on single-stranded
> phage. Thank you. Please respond to me by email to the address below.
Emily,
One thing you might try is to attempt second strand synthesis on the ssDNA
invitro. Take your DNA template and anneal random hexamers at room
temperature. Follow this by extension using Klenow fragment or T4 DNA
polymerase in the presence of a radioactive dNTP(alpha 32P). Do a dsDNA
control like pUC19 in parallel. If your DNA is single-stranded it should
generate a high amount of TCA-precipitable DNA using this method whereas a
dsDNA control should have very poor levels of incorporation because the
random hexamers are not able to penetrate the base-paired strands. If you
heat-denature your dsDNA control before the annealing of the hexamers the
TCA-precipitable counts should go up indicating the presence of single
stranded regions.
Another test is to heat-denature/snap cool the DNA prior to loading on a
neutral agarose gel and compare the migration of the sample with an
undenatured aliquot. If the bacteriophage DNA is single-stranded there is
unlikely to be any drastic change in electrophoretic mobility with or
without denaturation. On the other hand, heat-denatured dsDNA (linear or
covalently-closed circular) will migrate quite differently compared to its
undenatured counterpart.
Just some quick experiments which came to mind.
Oh...you might wish to look for a double stranded replicative
intermediates of your phage within infected cells a la the M13 phage
(check the lab techniques "bible" by Maniatis/Sambrook for more
information on this).
Cheers
Karl the hepB guy
posted and mailed